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Liang et al. Radiation Oncology 2012, 7:213http://www.ro-journal.com/content/7/1/213

RESEARCH Open Access

Autophagy inhibition plays the synergetic killingroles with radiation in the multi-drug resistantSKVCR ovarian cancer cellsBing Liang1, Dejuan Kong1, Yang Liu1, Nan Liang1, Mengzi He1, Shumei Ma1* and Xiaodong Liu2*

Abstract

Purpose: Autophagy has attracted attentions as a novel mechanism for tumor development. In this study Humanovarian carcinoma cell line SKOV3 and multidrug-resistant phenotype SKVCR cells were used and the roles ofautophagy in radiation-induced cell death were analyzed.

Methods and materials: Cell viability was examined by colony formation and cell counting kit-8 (CCK-8) assay,3MA and ZVAD were used to block autophagy and apoptosis, respectively. Quantitative real-time PCR was used todetect mRNA level and Western blot was used to detect protein expression, monodansylcadaverine (MDC) stainingand flow cytometery were used for autophagy, apoptosis and cell cycle dynamics, respectively.

Results: (1) The radiosensitivity exhibited differently in SKOV3 and SKVCR cells (SKOV3: D0=3.37, SKVCR: D0= 4.18);compared with SKOV3 the constitutive expression of MAPLC3 in SKVCR was higher, but no change of Caspase-3 andcleaved Caspase-3. (2) The ionizing radiation (IR)- induced apoptosis and autophagy were significant in both cells (P<0.05);inhibition of apoptosis with ZVAD showed no impact on survival of SKOV3 and SKVCR cells after radiation, whileinhibition of autophagy significantly decreased viability in SKVCR cells, for SKVO3 cells only low level of radiation (2 Gyand 4 Gy) could decrease the viability(P<0.05). (3) ZVAD inhibited apoptosis and autophagy in both cells, 3MA inhibitapoptosis in SKOV3, and promote apoptosis in SKVCR, together with inhibition of autophagy. (4) G2/M arrest was inducedby radiation in both cells; the accumulation of G2/M was more significant in SKOV3, 3MA attenuated theradiation-induced S phase delay in SKVCR.

Conclusion: IR-induced autophagy provides a self-protective mechanism against radiotherapy in SKVCR cells, the use ofautophagy inhibitor, 3MA, increases the killing effects of radiation by inhibiting autophagy and radiation- induced S phasedelay, also by the increase of apoptosis, which suggests a better therapeutic strategy in drug- resistant SKVCR ovariancancer cells.

Keywords: Autophagy, Radiosensitivity, Multidrug resistance, Ovarian cancer, Apoptosis

IntroductionEpithelial ovarian cancer(EOC)is the most lethal Gynecolo-gic malignancy, with 21,990 estimated new cases and 15,460 deaths in USA in 2011[1]. Platinum/paclitaxel-basedchemotherapy is the current standard of treatment aftersurgical staging and resection of abdominal and pelvic can-cers. Despite the advances in chemotherapy, the prognosis

* Correspondence: [email protected]; [email protected] Laboratory of Radiobiology (Ministry of Health), School of Public Health,Jilin University, 1163 Xinmin Street, Changchun, Jilin 130021, China2Key Laboratory of Radiobiology (Ministry of Health), School of Public Health,Jilin University, Department of Radiology and Radiation Oncology,China-Japan Union Hospital, Changchun 130021, China

© 2012 Liang et al.; licensee BioMed Central LCommons Attribution License (http://creativecreproduction in any medium, provided the or

still remains poor since many patients develop abdominalor pelvic recurrence that is resistant to further chemother-apy. Radiotherapy has been shown to produce a responsein chemo-resistant ovarian cancers, and may offer the pos-sibility of improved tumor control[2].Tumor cells have the capacity to respond to chemo-

therapy and radiation through multiple growth arrestand cell death pathways [3-5]. Various modes of celldeath have been known, such as necrosis, apoptosis andautophagic cell death [6-8].Apoptosis, the type I programmed cell death, has been

widely investigated under different circumstance includ-ing radiotherapy and the one of the most important

td. This is an Open Access article distributed under the terms of the Creativeommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.

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Liang et al. Radiation Oncology 2012, 7:213 of 12http://www.ro-journal.com/content/7/1/213

strategies for cancer treatment over the past decades.However, apoptosis is not the predominant form of celldeath as we have predicted, accounting for only 20% ofcases[9]. While, autophagy, the type II programmed celldeath, has been recently reported to play roles in the de-velopment of cancer. Autophagy is an evolutionarilyconserved catabolic process for the degradation and re-cycling of cytosolic, long-lived, or aggregated proteins,and excess or defective organelles, and is primarily a re-sponse to the stress of irradiation [10], chemotherapeuticagents [7], starvation [11,12], growth factors withdrawal[13], hypoxia or viral infection [14-17]. Dual roles ofautophagy have been reported, promotion of cell survivalor leading to cell death [18,19]. Therefore, autophagy isconsidered to be a double-edged sword in the process oftumor development. Elucidation of autophagy roles intreatment responsiveness at different stages of cancerprogression is a complex and challenging task. Betterunderstanding of autophagy regulation and its impact ontreatment outcomes will potentially provide novel thera-peutic targets in cancer.In this study, the human ovarian cancer cell line

SKOV3 and multidrug-resistant phenotype SKVCR cellswere used and the killing effect of different radiation for-mulae was assessed, the inhibitors of autophagy andapoptosis were used to explore the synergetic effect andthe potential mechanism. These results will contributeto improve the treatment efficacy for radiation-resistantMDR phenotype ovarian cancer and bring new insightsfor cancer development.

Materials and methodsCell cultureHuman ovarian carcinoma cell lines SKOV3 andmultidrug-resistant phenotype SKVCR cells were ob-tained from British Columbia Cancer Research Centre,Vancouver, BC, Canada. Cells were maintained inα-MEM with 10% fetal bovine serum and 100 U/ml ofpenicillin/streptomycin, at 37°C in humidified atmos-phere containing 5% CO2. SKVCR cells were cultured inα-MEM medium containing 2.0μg/ml vincristine (VCR),to maintain the drug-resistant phenotype.

Irradiation and chemicalsDifferent radiation formulae were introduced as follows:(A) 0Gy: no treatment was given; (B) 2Gy×5: 2 Gy inevery day for 5 times; (C) 1Gy ×2 ×5: 2 Gy (1 Gy 2 timesa day, 4 h interval) in every day for 5 times; (D) 10Gy×1:10 Gy for 1 time. ZVAD (20μM, Enzo, USA) and 3MA(2.5 mM or 5 mM Sigma-Aldrich Inc, USA) were usedas inhibitor of apoptosis and autophagy, respectively.These agents were administered into cells 2 h before ra-diation.X-ray irradiation was performed by using 180-

KVp X-ray generator (Model XSZ-Z20/20, China) at adose rate of 0.40Gy/min.

CCK-8 assayRadiosensitivity of SKOV3 and SKVCR was assayed byusing a Cell Counting kit-8 (CCK-8, Dojin Laboratories,Kumamoto, Japan). 1×104 cells/well were seeded in 96-well plates containing complete medium and incubatedfor 24 h followed by different doses of ionizing radiationfor 48h. 10 μl of the CCK-8 solution was added to eachwell and went on with incubation for 2 h in incubator.The absorbance was measured at 450 nm using a micro-plate reader (Synergy HT, Bio-Tek, USA).

Colony formation assayAfter the treatment of radiation, cells were plated into60mm petri dishes using standard culture media. Twoweeks later, cells were fixed with 4% formaldehyde,stained with crystal violet and colonies containing morethat 50 cells were counted and normalized to their cor-responding non-irradiated control. The surviving frac-tion for a given treatment was calculated as the platingefficiency of the irradiated samples relative to that of thesham-irradiated ones. For each dose level in four groups,three independent experiments were done. Multi-targetclick model of GraphPad Prism 5.0 (Systat Software,USA) was used to fit cell survival curves. The dose qua-sithreshold (Dq) and mean lethal dose (D0) werecalculated.

Quantitative real-time PCRTotal RNA was isolated using RNAiso Plus (Takara Co.Japan) according to the manufactures’ instructions.Quality and quantity of RNA was analyzed by measuringthe A260/A280 ratio with ultraviolet spectrophotometry.Reverse transcription was performed with 2 μg of totalRNA using PrimeScript Rtreagent Kit (Takara Co,Japan). Then each sample was analyzed by quantitativereal-time PCR (qPCR) (Stratagene MX3000P, Japan) inthe SYBR Premix Ex TapII (Takara Co, Japan),setting thecycles as follows:10s/95°C PCR initial activation step; 40cycles of denaturation for 20 s/95°C and annealing stepfor 20 s/60°C. The change in mRNA levels was deter-mined by the formula 2−(ΔΔCT), where ΔCT is thevalue from the threshold cycle (CT) of the treated sam-ple subtracted from the CT value of untreated or zerotime-point control sample. The relative amount ofmRNA in the sample was normalized to GAPDHmRNA. The primers for genes were as follows: MAPL-C3II forward: 50-CCTAGAA GGCGCTTACAGCT-30 andreverse 50-GGGACAATTTCATCCCGAAC-30; Caspase 3forward: 50-TTCAGGCCTGCCGTGGTACA −3 and re-verse: 50-CCAAGAATAATA ACCAGGTGCT-3;GAPDH


forward: 50-GCACCGTCA A GGCTGAGAAC-30 andreverse 50- TGGTGA AGA CG CCAGTGGA-30.

Western blot analysisAfter the indicated treatment, 40 μg of total protein wasseparated by SDS-PAGE, using a gradient gel [(10–12%),Bio-Rad Laboratories], transferred to nitrocellulose mem-brane, and analyzed by immunoblotting using the che-miluminescence (Santa Cruz, CA, USA). The primaryantibodies used were Caspase 3 (Cell Signaling, Beverly,MA, USA,1:500), MAPLC3 (Cell Signaling, Beverly, MA,USA,1:500) or GAPDH (Santa Cruz, CA, USA,1:1000),peroxidase-conjugated anti-mouse IgG or peroxidase-conjugated anti-rabbit IgG. (Santa Cruz, CA, USA, 1:1000).

Figure 1 The radiosensitivity in Human ovarian carcinoma cell lines Sformation assay was used to detect the survival curve, and Multi-target cliccell survival curves. SKOV3: D0= 3.37,Dq=0.60, n=1.508; SKVCR: D0= 4.18, Dqradiation formulae (0Gy,1Gy×2×5,2Gy×5,10Gy×1), cell viability was analyzedSKVCR cells by CCK-8 assay. (D) SKOV3 and SKVCR cells were exposed to diwas analyzed by CCK-8 assay. *P<0.05, vs sham-irradiated.

The intensity of protein bands were quantified usingimage j software and the ratio of specific band to controlwas analyzed.

MDC assay for visualization of autophagic vacuolesCells were seeded on cover slips over night followed bytreatment with different doses of radiation, 48h laterautophagic vacuoles were labeled with monodansylcada-verine (MDC) by incubating cells with 50 μM MDC inα-MEM at 37°C for 1 h. After incubation, cells werewashed with PBS and fixed with a solution of 4% paraf-ormaldehyde for 20 min. Autophagic vacuoles wereexamined using a fluorescence microscopy (Olympus,XSZ-D2).

KOV3 and multidrug-resistant phenotype SKVCR cells. (A) Colonyk model of GraphPad Prism 5.0 (Systat Software, USA) was used to fit=1.05, n=1.78. (B) SKOV3 and SKVCR cells were exposed to differentby the Colony formation assay. (C) Dose-effects analysis in SKOV3 andfferent radiation formulae (0Gy,1Gy×2×5, 2Gy×5, 10Gy×1 ), cell viability


Flowcytometry analysisCells were collected 24 h after radiation and 5 × 105

of cells were used for each sample. For cell cycle dis-tribution analysis, cells were stained with RNase -con-taining PI (propidium iodide) solution. For apoptosisdetection, cells were stained with PI and FITC labeledAnnexin-V. Stained cells were detected by Flow Cyto-metry (BD Biosystems, USA) and data were analyzedwith CellQuest (BD Biosciences) and FlowJo softwares(Tree Star Inc.).

Statistical analysisAll experiments were performed thrice and the datawere expressed as means ± SD. The difference be-tween two mean values was evaluated by using theStudent’s t -test and considered to be statistically sig-nificant when P < 0.05.

ResultsThe radiosensitivity in human ovarian carcinoma SKOV3and multidrug-resistant phenotype SKVCR cellsSKOV3 and SKVCR cells were exposed to various dosesof radiation (0, 2, 4, 6 or 8 Gy), cell viability was mea-sured by Colony formation and CCK8 assay. As shownin Figure 1A and C, radiation decreased the cell viabilityin SKOV3 and SKVCR, the D0, Dq and N values wereas follows: for SKOV3, D0= 3.37, Dq=0.60, n=1.508; forSKVCR, D0= 4.18, Dq=1.05, n=1.78. SKOV3 and

Figure 2 The endogenous expression of MAPLC3 and Caspase-3 in SKlevel of MAPLC3. (B) Western blotting was used to detect the expression oCaspase-3. (D) Western blotting was used to detect the expression of Casp

SKVCR cells were than exposed to different radiationformulae(0Gy, 1Gy×2×5, 2Gy×5,10Gy×1)and cell viabil-ity was analyzed by the Colony formation and CCK8assay. As shown in Figure 1B and D, radiation decreasedthe cell viability in SKOV3 and SKVCR, there was nodifference among different radiation treatment. Theseresults showed that SKOV3 and SKVCR cells exhibiteddifferences in terms of radiosensitivity, radiation sup-pressed the survival fraction more significantly inSKOV3 than in SKVCR (P < 0.05).

The endogenous expression of MAPLC3 and Caspase-3 inSKOV3 and SKVCR cellsAfter we found a variation in the radiosensitivity be-tween the two cell lines, we sought to find the contri-butions of apoptosis and autophagy to the total celldeath. The basal autophagic and apoptotic levels weremeasured, Figure 2 shown the expression level ofMAPLC3 II and Caspase-3 at the mRNA and proteinlevel which correlates with the autophagosome forma-tion and apoptosis, respectively, the higher constitutiveexpression of MAPLC3 II was detected in SKVCR byreal-time PCR (RT-PCR) and Western blot comparedwith SKOV3 cells. However, we did not find significantdifference in the mRNA and protein level of bothCaspase-3 and cleaved Caspase-3 (marker of apoptosis)between the two cell lines (Figure 2C and D).

OV3 and SKVCR. (A) Real-time RT-PCR was used to detect the mRNAf MAPLC3. (C) Real-time RT-PCR was used to detect mRNA level ofase-3. GAPDH was used as internal control.

Figure 3 (See legend on next page.)


(See figure on previous page.)Figure 3 The changes in apoptosis and autophagy after the treatment of irradiation in SKOV3 and SKVCR. (A,C) Flow cytometry was useto quantitative the apoptotic rate in SKOV3 and SKVCR. (B,D) Statistical analysis of apoptotic rate. Results were expressed as the percentage ofuntreated cells, Mean ± SD of thrice. *P<0.05, vs sham-irradiated. (F,H) MDC staining was used to detect the morphologic changes of autophagyin SKOV3 and SKVCR. (G,I) Statistical analysis of autophagic rate based on MDC staining, Mean ± SD of ten vision fields. *P<0.05, vs sham-irradiated. (J) Western blot was used to detect MAPLC3, the increase of MAPLC3II suggested the autophagy occurrence.


Ionizing radiation- induced apoptosis and autophagy inboth ovarian cancer cellsTo understand the underlying mechanism of radiation-induced cytotoxicity in SKOV3 and SKVCR cells, wemanaged to explore the roles of apoptosis and autop-hagy after radiation. Flow cytometric analysis was usedto detect apoptosis and MDC stain and western blotwere used to detect autophagy. In SKOV3 cells thebasal apoptotic rate in sham-irradiated group (control)was 4.6%, exposure of cells to the different therapeuticregimens of IR significantly increased the apoptosis(Figure 3A and B). Similar scenario was exhibited inSKVCR cells (Figure 3C and D), exposure to the differ-ent regimens of IR enhanced the autophagic activitysignificantly in both cell lines as detected by MDC im-munofluorescence and increased MAPLC3II/I ratio(Figure 3F-J).

Enhancement of radiation-induced cytotoxicity byblocking autophagy in SKVCR cellsThe role of autophagy during cancer therapy is paradox-ical and seems to be dependent on cell context. Thus,3MA and ZVAD were used to block autophagy andapoptosis respectively. SKOV3 and SKVCR cells wereexposed to different dose of radiation (0 Gy,2 Gy,4 Gy,6Gy,8 Gy) or different radiation formulae (0 Gy,1 Gy×2×5,2Gy×5,10 Gy×1) with or without 3MA (2.5 mM or 5 mM)and ZVAD(20 μM), cell viability was analyzed by CCK-8assay. As shown in (Figure 4), inhibition of autophagysignificantly reduced the cell viability of both cell lines.Importantly, In SKVCR cells, 3MA significantlyimproved the IR-induced cytotoxicity. In SKOV3 cellsthe IR- induced cell death was significantly increased by3MA in 2 Gy and 4 Gy groups (P <0.05). The IR-induced cell death was significantly increased by 3MAin SKVCR cells (P<0.05), but no change in SKOV3 cells(P>0.05). These findings indicate that autophagy plays apredominant pro-survival role in MDR-ovarian cancercells, SKVCR.

Transaction between autophagy and apoptosis in ovariancancer cellsNext we wanted to simply delineate the underlyingmechanism behind the enhanced radiosensitivity uponinhibition of autophagy. Therefore, 3MA and ZVADwere used to block autophagy and apoptosis respectively.

Flow cytometry analysis was used to detect apoptosis,MDC stain and western blot were used to detect autop-hagy Pre-treatment of SKOV3 and SKVCR with ZVADbefore exposure to IR markedly reduced the IR stimula-tory effect on apoptosis. Pre-culture of SKOV3 cells with3MA, significantly attenuated the IR-induced apoptosis.Importantly, in SKVCR, the apoptotic rate showed sig-nificant elevation upon pre-treatment with 3MA in irra-diated and non-irradiated cells. We also found theincrease of cleaved caspase-3(apoptosis marker) inSKVCR cells, wherein autophagy inhibition increasedthe expression of cleaved caspase-3 before and after ra-diation exposure (Figure 5A-E). Blocking autophagy with3MA in SKVO3 and SKVCR significantly reduced thenumber of cells undergoing autophagy as estimated byMDC staining. Importantly, pre-culture of both cell lineswith ZVAD attenuated the IR-induced autophagic re-sponse (Figure 5F-I). These findings indicate the pres-ence of crosstalk between apoptosis and autophagy.More importantly, the enhanced radiosensitivity inSKVCR cells upon suppressing autophagy could be dueto concomitant up-regulation of apoptosis.

Autophagy inhibition alters the IR-induced cell cyclearrest in SKVO3 and SKVCR cellsFinally, we sought to explore whether autophagy changeswas associated with cell cycle regulation. Hence, flowcytometry analysis was performed pre- and post-IR inthe presence or absence of 3MA. In SKOV3 cells, radi-ation decreased the cell number in G1/S phase signifi-cantly and induced accumulation of cells at G2/Mphase. Inhibition of autophagy in SKOV3 cells with3MA for 24 hours prior to irradiation caused borderlineascend in G1/S phase, while G2/M phase also decreasedsignificantly (2.5 fold), compared with IR alone group(Figure 6A-D). In SKVCR cells, radiation decreased thecell number in G1/S phase significantly and induced ac-cumulation of cells at G2/M phase, but S phase delaywas also observed. When the cells were pretreatedwith 3MA, the cell number in G1/S phase increasedslightly, while S phase showed significant reduction afterradiation as compared with radiation alone group(Figure 6E-H). These results suggest that attenuation ofthe IR-induced S phase delay by 3MA could be also onecontributory factor in the enhanced radiosensitivity uponautophagy inhibition.

Figure 4 Effects of inhibitors for autophagy and apoptosis on the cell survival rate. (A) Dose-effect changes after radiation in SKVO3 cellswith or without 3MA(2.5mM) and ZVAD(20μM). (B) Dose-effect changes after radiation in SKVCR cells with or without 3MA(2.5mM) and ZVAD(20μM). (C) SKOV3 cells were exposed to different radiation formulae (0Gy,1Gy×2×5,2Gy×5,10Gy×1) with or without 3MA (5mM) and ZVAD(20μM),cell viability was analyzed by CCK-8 assay. (D) SKVCR cells were exposed to different radiation formulae (0Gy,1Gy×2×5,2Gy×5,10Gy×1) cells with orwithout 3MA (5mM) and ZVAD(20μM), cell viability was analyzed by CCK-8 assay. *P<0.05, vs sham-irradiated. Results were expressed as thepercentage of untreated cells, Mean ± SD of thrice. *P<0.05, vs untreated cell.


DiscussionOvarian cancer is one of the most common genitalmalignant tumors for female, maximal cytoreductivesurgery together with platinum-based chemotherapyis the initial treatment program [20]. Drug resistanceis a major obstacle to successful treatment of ovariancancers [21]. Therefore, overcoming of drug-resistancein ovarian cancer would become an important factor toimprove treatment efficacy. Radiotherapy functions as anadjuvant therapy for the treatment of ovarian cancer[22-24], mainly applies to the pre-operative and post-operative adjuvant therapy or palliative treatment ofadvanced ovarian cancer [25,26]. Radiotherapy has beenshown to produce a response in chemo-resistant ovarian

cancers, and may offer the possibility of improved tumorcontrol. Radiosensitivity of malignant ovarian tumorsvaries markedly, dysgerminoma was classified as highlyradiosensitive, while ovarian endodermal sinus tumor,emmature teratoma and embryonal carcinoma showedthe lowest radiosensitivity. Epithelial ovarian cancer(EOC) and eranular cell carcinoma are moderatelyradiosensitive.To improve the efficacy of treatment of drug-resistant

ovarian cancer, the human epithelial ovarian cancer cellline SKOV3 and multidrug-resistant(mdr) phenotypeSKVCR cells were exposed to different dose of radiation(0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy) or different radiation for-mulae (0 Gy, 1 Gy×2×5, 2 Gy×5, 10 Gy×1) with or

(See figure on previous page.)Figure 5 The interrelationship between autophagy and apoptosis in SKOV3 and SKVCR. Apoptosis and autophagy were determined in thepresence or absence of 3MA or ZVAD, with or withour indicated IR regimens at 24 h. (A, C) Flow cytometry was used to quantitative theapoptotic rate in SKOV3 and SKVCR. (B, D) Statistical analysis of apoptotic rate Results were expressed as the percentage of untreated cells, Mean± SD of thrice. *P<0.05, vs untreated cells. (E) Western blot was used to detect caspase 3 and cleaved caspase 3 in SKVCR, the increase of cleavedcaspase 3 suggested the apoptosis occurrence. (F,H) MDC staining was used to detect the morphologic changes of autophagy in SKOV3 and SKVCR.(G,I) Statistical analysis of autophagic rate, results were expressed as the percentage of untreated cells, Mean ± SD of thrice. *P<0.05, vs untreated cells.


without 3MA(autophagy Inhibitor via PI3K regulation)and ZVAD (apoptosis Inhibitor via caspase-3 regulation),the changes of cell viability, apoptosis, autophagy andcell cycle were analyzed in this study.In order to know the radiosensitivity of ovarian can-

cers, cell viability was measured by colony formationand CCK8 assay, radiation could induce cell death inboth cell lines and the mdr-phenotype SKVCR showedmore resistant than SKVO3. In general, the radiosensi-tivity is proportional to the proliferation rate. One previ-ous study found that SKOV3 and SKVCR showeddifferent cell doubling time, the doubling time in SKVCRcells was prolonged approximately 1.8 times (53 hoursfor SKVCR and 29 hours for SKOV3 cells) [27]. Zhanget al. reported the mdr -transfected K-562 cells showedresistance to radiation as compared with parental cells[28], a similar trend was reported by Wei R et al. [29].Together with our data, the fact that multi-drug resist-ant cells are less radiosensitive as compared with paren-tal cells has been further confirmed. How to treat thiskind of drug resistant and radiation-resistant cancer hasbeen the hot point.Next we managed to find what’s the difference and the

underline mechanism which make SKVCR cells resistantto chemotherapy, this will contribute to find more targetfor treatment. It is well-known that apoptosis is import-ant in determining the outcome of chemo- andradiation- therapy [30,31] and can be triggered by antic-ancer drugs and radiation. Besides, mounting evidencesuggest that cancer cells can commit to death by variousnon-apoptotic pathways such as autophagy [32]. Thenwe detected the constitutive expression of different typesof cell death and found that the basal level of autophagyin SKVCR was significantly higher than in SKOV3 cells,but for apoptosis there was no difference betweenSKVCR and SKOV3 cells, suggesting autophagy mightassociate with the MDR characteristics and play a pro-survival role. Autophagy is a highly conserved cellularprocess. It involves degradation of intracellular orga-nelles and long-lived proteins to yield amino acids reuseand energy recycle [33]. It was demonstrated in previousresearches that autophagy can have either pro-survivalor pro-death functions in an organism, depending on itslevel of activation [34-37].To make sure which type of cell death, autophagy or

apoptosis, would play predominant roles in killing effect

of radiation, 3MA and ZVAD were used to block autop-hagy and apoptosis respectively. Our result showed thatalthough these two cell lines were different in terms ofbasal autophagy, ionizing radiation can induce both ofthem to undergo apoptosis and autophagy (Figure 3). In-hibition of apoptosis with ZVAD showed no impact onsurvival of SKOV3 and SKVCR cell lines after radiation,while inhibition of autophagy significantly decreased via-bility in SKVCR cells, for SKVO3 cells only low level ofradiation (2 Gy and 4 Gy) could decrease the viability(Figure 4), suggesting autophagy might be the predomin-ant mechanism after radiation rather than apoptosis.Moreover, the complex interrelationship between

apoptosis and autophagy has been reported to beaffected by various biochemical processes via differentpathways [38-40], and growing number of studies sug-gested the presence of crosstalk between autophagy andapoptosis; (a) Autophagy may be indispensable for apop-tosis occurrence and lead to cell death; (b) Autophagymay antagonize apoptosis and make cells survive fromstimuli; (c) Apoptosis and autophagy may occur inde-pendent of each other, there might be molecular switchbetween them, both autophagy and apoptosis determinethe final fate of cells [41]. In this study ZVAD inhibitapoptosis and concomitantly inhibited autophagy in bothcell line (Figure 5). Wu YT also got the same results andfound ZVAD could inhibit lysosomal enzyme cathepsinB activity, and subsequently blocked autophagosomematuration [42]. Interestingly, 3MA play a different ef-fect on apopotosis in SKOV3 and SKVCR, e.g., 3MA in-hibit apoptosis in SKOV3, and promote apoptosis inSKVCR (Figure 5), together with inhibition of autophagy.Due to direct inhibition of autophagy and apoptosis ac-tivity by 3MA in SKOV3 cells, the pro-survival role ofautophagy together with the death outcome of apoptosisattenuated the radiation-induced cell death to a signifi-cant extent. While in SKVCR cells autophagy inhibitiontriggered the up-regulation of apoptosis, suggestingthere might be a molecular switch between autophagyand apoptosis in SKVCR cells, autophagy coordinatedwith apoptosis to improve the radiosensitivity. In recentyears, more and more researches suggested that theapoptosis and autophagy could be mutually antagonisticor promoted in some scenario. The same induction fac-tors can play a positive or negative role for the two kindsof programmed cell death in different cells. For example,

(See figure on previous page.)Figure 6 Autophagy inhibition attenuates IR-induced cell cycle arrest. (A) Flow cytometry was used to quantitative the cell cycle rate withor without autophagy inhibitor, 3MA (5 mM), with or without indicated IR regimens at 24 h in SKVO3; SKOV3 cell population (%) in G1 phase (B),S phase (C), G2 phase (D) were quantified. (E) Flow cytometry was use to quantitative the cell cycle rate with or without autophagy inhibitor,3MA (5mM, with or without indicated IR regimens at 24 h in SKVCR. SKVCR cells were treated with IR or a combination of both (5 mM 3MAfollowed by IR) for 24 h, SKVCR cell population (%) in G1 phase (F), S phase (G), G2 phase (H) were quantified. All data are representative of threeindependent experiments and are shown as the mean ± SD. *P<0.05. vs untreated 3MA group.


3MA could inhibit autophagy at the same time promoteor inhibit apoptosis [43]. Which has been confirmed byour data.In general, it has been found that cell radiosensitivity

is directly proportional to the rate of cell division and in-versely proportional to the degree of cell differentiation,this is described by the law of Bergonie and Tribondeau,formulated in 1906 [44]. With regard to relation be-tween cell cycle and radiosensitivity, cells are least sensi-tive when arrested in the S phase, then the G1 and G2phase, the most sensitive one is the M phase of the cellcycle. Ge JN et al. showed that starvation could not onlyinduce autophagy in tumor cells, but also caused tumorcell cycle arrest [45]. Here, we showed that G2 / M phasearrest was induced by radiation in SKOV3 cell andSKVCR cell lines. The accumulation of G2 / M phasecells was more significant in SKOV3 cells. These resultsgo along with many previous reports, for example,Rui Wei et al. found radiation with 6 MV X-rays causedG2/M arrest A549 and A549/DDP cells [29]. In ourstudy, 3MA could inhibit autophagy and attenuate theradiation-induced S phase delay in SKVCR, suggesting3MA regulate cell cycle by redistribution, adjustingradiation-induced cell cycle redistribution. Therefore,3MA synergized the killing effects of radiation by de-creasing the proportion of cells in S phase which hasbeen thought the least sensitive phase to radiation, con-sequently increased the radiosensitivity of SKVCR cells.

ConclusionsThe 3MA can not only inhibit autophagy, but also in-hibit the radiation- induced S phase delay and increaseapoptosis in SKVCR cells, ultimately leading to the in-crease of cell death. These data illustrated that inhibitionof autophagy may enhance the cell-killing effect ofradiotherapy in multidrug-resistant human ovarian can-cer cells, which may represent a novel approach to in-crease the efficacy of radiotherapy as an anticancermodality.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsLB completed most of the experiments and drafted the manuscript. KDJ, LY,LN and HMZ performed colony formation, flowcytometry analysis andMAPLC3II detection. LXD and MSM participated importantly in the

conception and design and helped to finished the manuscript. All authorsread and approved the final manuscript.

Received: 31 May 2012 Accepted: 11 December 2012Published: 17 December 2012

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doi:10.1186/1748-717X-7-213Cite this article as: Liang et al.: Autophagy inhibition plays thesynergetic killing roles with radiation in the multi-drug resistant SKVCRovarian cancer cells. Radiation Oncology 2012 7:213.

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